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Intervals to Plasmodium falciparum recurrence after anti-malarial treatment in

pregnancy: a longitudinal prospective cohort
Malaria Journal Sample
(2015) 14:221
doi:10.1186/s12936-015-0745-9
Natthapon Laochan (natthapon@shoklo-unit.com)
Sophie G. Zaloumis (sophiez@unimelb.edu.au)
Mallika Imwong (Mallika@tropmedres.ac)
Usa Lek-Uthai (usa.lek@mahidol.ac.th)
Alan Brockman (albrockman64@gmail.com)
Kanlaya Sriprawat (poo@shoklo-unit.com)
Jacher Wiladphaingern (sam@shoklo-unit.com)
Nicholas J. White (nickw@tropmedres.ac)
Franois Nosten (francois@tropmedres.ac)
Rose McGready (rose@shoklo-unit.com)
Sample

ISSN
Article type

1475-2875
Research

Submission date

6 April 2015

Acceptance date

21 May 2015

Article URL

http://dx.doi.org/10.1186/s12936-015-0745-9

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2015 Laochan et al.

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Intervals to Plasmodium falciparum recurrence after

anti-malarial treatment in pregnancy: a longitudinal
prospective cohort
Natthapon Laochan1
Email: natthapon@shoklo-unit.com
Sophie G. Zaloumis2
Email: sophiez@unimelb.edu.au
Mallika Imwong3,4
Email: Mallika@tropmedres.ac
Usa Lek-Uthai5
Email: usa.lek@mahidol.ac.th
Alan Brockman1
Email: albrockman64@gmail.com
Kanlaya Sriprawat1
Email: poo@shoklo-unit.com
Jacher Wiladphaingern1
Email: sam@shoklo-unit.com
Nicholas J. White3,4
Email: nickw@tropmedres.ac
Franois Nosten1,3,4
Email: francois@tropmedres.ac
Rose McGready1,3,4*
*
Corresponding author
Email: rose@shoklo-unit.com
1

Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research

Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand
2

Centre for Epidemiology and Biostatistics, Melbourne School of Population and

Health, University of Melbourne, Melbourne, Australia
3

Mahidol-Oxford Tropical Medicine Research Unit (MORU), Faculty of

Tropical Medicine, Mahidol University, Bangkok, Thailand
4

Centre for Tropical Medicine and Global Health, Nuffield Department of

Medicine, University of Oxford, Oxford, UK

Department of Parasitology and Entomology, Faculty of Public Health, Mahidol

University, Bangkok, Thailand

Abstract
Background
Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment
failure is common. The objective of this study was to examine the duration of persistent
parasite carriage following anti-malarial treatment in pregnancy.

Methods
The data presented here are a collation from previous studies carried out since 1994 in the
Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed
using the same unique methodology detailed in the Materials and Methods section. Screening
for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and
therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women
with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick
sample collected. Parasite DNA was extracted from the blood-spot samples using saponin
lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2
and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or
indeterminate. Factors associated with time to microscopy-detected recrudescence were
analysed using multivariable regression techniques.

Results
From December 1994 to November 2009, 700 women were treated for P. falciparum and
there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR
genotyping. Most of the recurrences, 85 % (770/909), occurred after treatment with quinine
monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean
number of days to recurrence was significantly shorter in women with recrudescent infection,
24.5 (95 %: 23.4-25.8), compared to re-infection, 49.7 (95 %: 46.9-52.7), P <0.001. The
proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63
from the start of treatment was 29.1 % (124/428), 13.3 % (57/428) and 5.6 % (24/428).
Recrudescent infections 100 days after treatment occurred with quinine and mefloquine
monotherapy, and quinine + clindamycin and artesunate + atovaquone-proguanil combination
therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax
infection increased the geometric mean interval to recrudescence by 1.28-fold (95 % CI:
1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased
0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence)
and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia.

Conclusions
Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial
treatment. Long intervals to recrudescence are more likely with the use of artemisinincontaining treatments and also observed with intercalated P. vivax infections treated with
chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration
of follow-up, preferably until delivery or day 63, whichever occurs last.

Background
In order to reduce malaria-related maternal mortality in a low-transmission area where
multidrug-resistant (MDR) strains of Plasmodium falciparum are prevalent, frequent
screening and treatment of all positive malaria episodes is required. This has been a focus
area of the antenatal clinics (ANCs) of Shoklo Malaria Research Unit (SMRU) on the
Thailand-Myanmar border [1, 2]. In 1994, PCR genotyping of P. falciparum parasite
recurrence was introduced to distinguish novel or new infections from recrudescent infections
[3]. This is the accepted method for reporting trials of anti-malarial drug efficacy [4, 5]. PCR
genotyping results of P. falciparum at SMRU have been published within the context of
treatment efficacy trials in pregnant women [611] and in non-pregnant patients [12, 13]. The
longest reported time to recrudescence confirmed by PCR genotyping in non-pregnant
patients is 62 days [3] but the duration of follow-up in P. falciparum anti-malarial drug trials
is limited to 63 days [4]. In pregnancy, when a woman may naturally follow antenatal care
for an extended period, much longer carriage times have been reported: 133 days in Malawi
[14], 187 days in Mozambique [15], 85 days [7], 98 days [10] and 121 days [3] on the
Thailand-Myanmar border.
In the era of malaria therapy from the 1920s to the 1950s, and the volunteer studies
conducted to assess new anti-malarial drugs conducted in Australia and the USA, natural
infections in previously non-immune subjects were observed to last for many months.
Transfusion malaria infections acquired from donors who had left the endemic area years
previously were also documented [16]. In the early 1950s, Eyles and Young [17] reported the
duration of incompletely treated, artificial P. falciparum infections (South Carolina strain) in
neurosyphilis patients. The average duration of infection was 222 days with three of the
infections persisting for more than one year, and the longest 480 days. In a similar report with
the Panama strain of P. falciparum there were 23 incompletely treated infections which
persisted for an average of 280 20 (range 114 to 503) days, with four cases persisting for
more than one year [18]. These reports occurred long before the PCR genotyping technique
was introduced [19, 20] but the concept of prolonged asymptomatic carriage of P. falciparum
is well established [21]. The longest recorded persistence of P. falciparum in an individual is
13 years [16]. Here P. falciparum genotyping was used to examine the duration of persistent
P. falciparum carriage in pregnant women and examine the factors associated with time to
recrudescence.

Methods
Study site and population
Pregnant women in this series attended the weekly ANC of SMRU on the northwestern
border of Thailand. In this hilly, forested area malaria transmission is low and seasonal
(estimated entomological inoculation rate <2) in the whole population, including pregnant
women [22]. Acquired immunity is poorly protective and severe malaria is common at all
ages, especially in pregnant women [22]. At the start of the research programme, maternal
mortality from falciparum malaria was high: estimated 1,000 maternal deaths per 100,000
live births [2]. Women are invited to come to the ANC as soon as they are aware of their
pregnancy. All women attending consultation are screened weekly for malaria as the only
effective strategy to prevent maternal death [1]. Prophylactic vitamins (ferrous sulphate, folic
acid and vitamin B1) are provided to all women attending for consultation. Haematocrit
levels are measured twice weekly and anaemic women (haematocrit <30 %) receive treatment
doses of ferrous sulphate and folic acid. In the case of severe symptomatic anaemia
(haematocrit <20 %) screened blood transfusions are provided.

Treatment of Plasmodium falciparum in pregnancy

This study spans 15 years during which treatment of falciparum malaria has changed from
monotherapy to artemisinin-based combination therapy (ACT) (Fig. 1). Quinine based
therapy has always been the first-line treatment for uncomplicated malaria in the first
trimester. Quinine was also used throughout pregnancy until artemisinins were recommended
in 2005 for routine first-line therapy in the second and third trimesters for uncomplicated
malaria. Quinine-clindamycin (a non-artemisinin based combination therapy) has been used
almost exclusively in the first trimester since its introduction in 2005.
Fig. 1 Study flow
Treatment doses for uncomplicated malaria infections were prescribed as follows: quinine
three times daily (10 mg salt/kg every eight hours, Government Pharmaceutical Organization)
for seven days; artesunate (outside the first trimester) once daily (usually 2 mg /kg) for seven
days (total dose 1016 mg/kg, Guilin, PRC); artemether-lumefantrine (artemether 20 mg and
lumefantrine 120 mg, Novartis, Basel, Switzerland): four tablets twice daily for three days,
mefloquine 25 mg/kg stat dose or as a split dose (15 mg/kg and 10 mg/kg on consecutive
days); dihydroartemisinin-piperaquine once daily for three days (DHA 3 mg/kg, PPQ 1517
mg/kg, Holley Pharm PRC); artesunate (4 mg/kg/day) combined to atovaquone-proguanil
(atovaquone 20 mg/kg/day and 8 mg/kg/d) for three days (Malarone, Glaxo-Wellcome,
Dartford, UK). Since 2007, clindamycin was combined with artesunate or quinine at a dose
of 5 mg/kg three times daily (in practice in most women this was 300 mg three times daily,
Siam Bheasach). Chloroquine (Government Pharmaceutical Organization, Thailand) was
given once a day for three days, with a dose of 10 mg base/kg/day for two days and 5 mg
base/kg/day on the third day, for Plasmodium vivax. Treatment administration was either
unsupervised, when a pregnant woman was not admitted for treatment, or supervised when
the initial doses or all of the doses were administered by a health worker and the pregnant
woman was observed for vomiting. Pregnant women who vomited within 30 min of their
dose were re-administered a full dose and those who vomited within 30 to 60 min of initial
dosing were re-administered half the dose.

Contrary to the changes in treatment over time there has been no change in the unique
methodology applied to pregnant women in this area including the screening by microscopy
for malaria offered at every antenatal visit during pregnancy, in the basic data collected at the
time of each malaria episode and in the way the sample is collected. At the time of each
malaria episode the date, body temperature, history of fever in the previous 48 h,
parasitaemia, haematocrit, gestational age of the pregnancy, and malaria history were
recorded. After microscopy diagnosis and before treatment, fresh blood from a finger-prick
sample was used to make three blood spots (approximately 30 l each) on a strip of 3 M filter
paper. This was dried overnight before DNA extraction the next day.

Definitions
Symptomatic malaria was defined as slide-confirmed parasitaemia with a history of fever in
the previous 48 h or a measured axillary or aural temperature 37.5 C [23]. Asymptomatic
malaria was defined as slide-confirmed parasitaemia without any history of fever and a
measured temperature <37.5 C. The gestational age at the time of the malaria episode was
determined by ultrasound [24], the Dubowitz newborn examination [25] or by the fundal
height formula developed for this population [26]. Treatments were classified as
monotherapy when given without any other anti-malarial. Mefloquine and piperaquine were
categorized as long-acting drugs, lumefantrine and atovaquone-proguanil as medium-acting
drugs and the remainder as short acting.
For recrudescence risk factor based analysis anti-malarial therapies were grouped into
artemisinin based: including ACT and artesunate monotherapy and non-artemisinin based
including combinations such as quinine and clindamycin or monotherapy such as mefloquine.

Plasmodium falciparum PCR genotyping

Plasmodium falciparum infection from consecutive malaria episodes during pregnancy was
analysed by using the three loci genotypes: MSP1, MSP2 and GLURP. Nested PCR was the
amplification strategy used to genotype P. falciparum and this is explained in detail
elsewhere [3, 27, 28]. A parasite infection with the same three-locus genotype pre- and posttreatment was considered a recrudescence. Infections which differed pre- and post-treatment
were denoted as novel (or new) infections. Infections that could not be classified as
recrudescent or new were denoted as indeterminate. Only patients who became microscopy
smear negative following treatment were included in the analysis. The time to the next patent
infection was calculated by the difference in days from the date treatment started until the
date of the first recurrence detected by microscopy with active weekly screening.

Statistical analysis
Continuous variables were described using median (range) and categorical variables using
frequency (percentage). The distribution of the number of days to recurrence of P. falciparum
infection was positively skewed (Fig. 2) and therefore natural logarithm (loge) transformed.
Linear regression models were fitted to the loge transformed outcome, and either geometric
means or ratios of geometric means were derived. Separate sub-group analyses were
performed for women with recrudescent infection(s) and women with novel infection(s). In
the multivariable linear regression analyses, a risk factor was deemed to be associated with
the loge transformed outcome if the p-value was below the nominal significance level of 0.1;
multi-collinearity between each of the covariates (i.e., risk factors) was assessed using

variance inflation factors (VIFs; all <2); and all estimates derived from the regression
analyses were adjusted for age (years), weight (kg), and study period categorized into four
three-year intervals (199497, 19982001, 200205, 200609). As resistance appears with
increasing years of drug use, e.g., artemisinin, and as the cohort was conducted over many
years with different numbers of women available in different years, four blocks were chosen
for a reasonably evenly distribution of women in each block, so time could be controlled for
in regression analysis. To account for the correlation between the number of days to
recurrence of multiple P. falciparum infections during a single pregnancy, robust standard
errors were calculated using the Huber-White sandwich estimator. Tests for linear trends
were performed by fitting a linear model to the outcome variable with the categorical
covariate treated as pseudo continuous. Proportions were compared using Pearsons Chisquared test. Data were analysed in Stata/IC version 11.1 (StataCorp, College Station, Texas,
USA).
Fig. 2 Frequency of PCR-confirmed novel (white square) and recrudescent (black circle)
Plasmodium falciparum infections for each follow-up week from day 7 of treatment

Ethics approval
The data presented here represent a collation of data from women enrolled in treatment trials
in pregnancy all previously published and approved by the Faculty of Tropical Medicine
Ethics Committee in Bangkok and in later years the Oxford Tropical Research Ethics
Committee [611, 29].

Results
From December 1994 to November 2009, there were 700 women with 1,647 episodes of P.
falciparum of which 19 were classified as indeterminate by PCR; seven failed to amplify; no
PCR spot was found for two episodes and pregnancies with episodes of non-sequential pairs
were removed, leaving 1,609 episodes for analysis in 700 women (Fig. 1). Most of the
women, 691 (76.0 %), experienced a single recurrence of P. falciparum during their
pregnancy, 164 (18.0 %) had two and 54 (6.0 %) experienced three or more recurrences
within the same pregnancy. The maximum number of consecutive P. falciparum episodes
within a single pregnancy was five and all four recurrent infections could be characterized by
PCR in two women. The demographic characteristics of the pregnant women at the time of
the primary infection are reported in Table 1.
Table 1 Distribution of variables measured in 700 pregnant women before treatment of the
primary Plasmodium falciparum infection
Characteristic
Na
Median [range] or %
Age (years)
700
24.5 [1445]
Maternal weight (kg)
692
47 [3078]
Haematocrit (%)
660
31 [1248]
Parasitaemia /L
699
3,216 [16.4-4,768]
Temperature (C)
642
36.8 [35.0-40.4]
Symptomatic
654
67.3
a
N, number of observations

Most of the recurrent infections, 85 % (770/909), occurred following treatment with one of
the three most commonly used regimens: quinine monotherapy, artesunate monotherapy or
artesunate-clindamycin, which are all rapidly eliminated seven-day treatments (Table 2). Not
all drugs were used for treatment across all years of the data collection period, for example,
mefloquine monotherapy was only administered from 1994 to 1997 and in a very small
number of women (Table 2). The mean gestational age at the time of treatment was lowest
for quinine as it is the drug of choice for first trimester (Table 2). The number of recurrent
infections classified as recrudescent or novel varied with the anti-malarial treatment provided
(Table 2).

Table 2 Estimated gestational age and time to recurrence of Plasmodium falciparum (Pf) for novel and recrudescent infections for each antimalarial treatment and including the distribution of intercalated Plasmodium vivax infections
EGAd (weeks)

Time to novel infection

(days)

Time to recrudescent infection (days)

Anti-malarial (action, years)a

Nb
Median [range] N
Median [range]
N
Median [range]
Quinine monotherapy (short, 19942006)
476
17 [336]
173
47 [7216]
303
21 [7126]
Quinine + clindamycin (short, 19982009)
29
10 [424]
22
84 [28201]
7
50 [21122]
Artesunate monotherapy (short, 19952008)
151
21 [437]
102
54 [8182]
49
22 [1199]
Artesunate + Clindamycin (short, 19982009)
143
23e [139]
121
49 [21158]
22
28 [1270]
Artemether + lumefantrine (medium, 20042006)
43
23 [1336]
24
36 [15140]
19
23 [1463]
Artesunate + Atovaquone + proguanil (medium, 19992005) 11
23 [1832]
8
67 [44101]
3
49 [35120]
Mefloquine monotherapy (long, 19941997)
22
26 [1238]
4
25 [944]
18
22 [7123]
DHAf + Piperaquine (long, 20062008)
29
20 [834]
23
63 [35180]
6
33 [2149]
Artesunate + Mefloquine (long, 19952008)
5
28 [0.4-39]
4
35 [2043]
1
21 []
a
action, reflects length of time anti-malarial remains in the body after administration; years, period in which treatment was administered at SMRU
b
N, number of recurrent Pf episodes
c
% (N), percentage and number of intercalated P. vivax episodes
d
EGA, estimated gestational age (weeks) at time of malaria episode
e
one EGA missing
f
DHA, dihydroartemisinin

% >28 days
25.4 (77/303)
57.1 (4/7)
30.6 (15/49)
45.5 (10/22)
31.6 (6/19)
100.0 (3/3)
27.8 (5/18)
66.7 (4/6)
0.0 (0/1)

Intercalated P. vivax
% >42 days
10.6 (32/303)
57.1 (4/7)
12.2 (6/49)
27.3 (6/22)
26.3 (5/19)
66.7 (2/3)
5.6 (1/18)
16.7 (1/6)
0.0 (0/1)

% >63 days
5.0 (15/303)
42.9 (3/7)
4.1 (2/49)
9.1 (2/22)
0.0 (0/19)
33.3 (1/3)
5.6 (1/18)
0.0 (0/6)
0.0 (0/1)

% (N)c
33.0 (58)
7.4 (13)
21.6 (38)
26.1 (46)
6.3 (11)
1.7 (3)
0 (0)
4.0 (7)
0 (0)

Time to recurrence in recrudescent and novel infections

The geometric mean number of days to recurrence was significantly shorter in women with
recrudescent infection, 24.5 (95 %: 23.4-25.8) days, compared to re-infection, 49.7 (95 %:
46.9-52.7) days, P <0.001 (Fig. 2). There was no statistically significant difference in the
geometric mean number of days to recurrence according to the episode number, i.e., for
recurrent episodes that were recrudescent infections, the time from the primary infection to
first recrudescence (N = 353) was 24 (95 % CI: 2326) days; first to the second
recrudescence (N = 55) was 25 (95 % CI: 2229) days; and, from the second to the third or
more recrudescence (N = 20) was 28 (95 % CI: 2235) days, P = 0.44. For recurrent episodes
that were novel infections, the observed times were 50 (95 % CI: 4653) days for primary to
first (N = 339); 51 (95 % CI: 4658) days first to second (N = 108); and, 45 (95 % CI: 3853)
days for second to third or more infection (N = 34), P = 0.38.

Time to recurrence with intercalated Plasmodium vivax infection

Of the 909 P. falciparum recurrences, 19.3 % (176) had an intercalated Plasmodium vivax
infection between the P. falciparum primary and recurrent episode. There were 145 recurrent
P. falciparum episodes with one intercalated P. vivax episode; and 23, seven and one with
recurrent P. falciparum episodes with two, three and five intercalated P. vivax episodes,
respectively. The proportion of recrudescent P. falciparum infections following an
intercalated P. vivax infection was significantly lower than those without an intercalated P.
vivax episode: 19.9 % (35/176) compared with 53.6 % (393/733), P <0.001. In both novel and
recrudescent infection, intercalated P. vivax infection resulted in a longer geometric mean
number of days to recurrence compared to those without intercalated P. vivax: 71 (95 % CI:
6578) versus 43 (95 % CI: 4046) days, and 49 (95 % CI: 4159) versus 23 (95 % CI: 22
24) (P <0.001 for both), respectively. The proportion of intercalated P. vivax infections was
lower with medium- and long-acting anti-malarial treatments (Table 2).

Time to recurrence according to anti-malarial treatment

The geometric mean number of days to recrudescent infection(s) for short- (N = 381),
medium- (N = 22) and long- (N = 7) acting anti-malarials was 24 (95 % CI: 2326), 28 (95 %
CI: 2533) and 33 (95 % CI: 2643) days (linear trend: P = 0.02), when mefloquine
monotherapy was excluded from the analysis (only used from 199497, but high grade
resistance was already established during this period). When mefloquine monotherapy was
included, the geometric mean number of days to recrudescent infection for long-acting antimalarials fell to 25 (95 % CI: 2032) days and the linear trend was no longer significant
(linear trend: P = 0.85). There was no evidence of a linear trend in number of days to novel
infection for short-, medium- and long-acting anti-malarials.

Recrudescence after 28, 42 and 63 days of follow-up in pregnancy

Nearly one third (29 % (124/428)) of recrudescent infection occurred after day 28 with 13.3
% (57/428) and 5.6 % (24/428) occurring after day 42 and 63, respectively. The proportion of
quinine treatments that were true recrudescent infections that would be wrongly classified as
treatment success, if follow-up ceased at day 28, day 42 and day 63 were 26.1 % (81/310),

11.6 % (36/310) and 5.8 % (18/310), respectively (Table 2). For artemisinin-based treatments
the respective proportions were 38.0 % (38/100), 20.0 % (20/100) and 5 % (5/100) (Table 2).

Factors associated with time to a recrudescent infection

For four anti-malarial treatments (quinine and mefloquine monotherapy, quinine +
clindamycin and artesunate + atovaquone + proguanil), women were observed to have
recrudescent infections 100 or more days from the start of drug treatment (Table 2). There
were no intercalated P. falciparum recrudescent infections (i.e., a recrudescence of a primary
infection after an intervening novel infection).
Estimated ratios of geometric means are presented in Table 3 for eight risk factors
hypothesized to be associated with the number of days to recrudescent P. falciparum
infection. Of the eight risk factors, anti-malarial therapy, therapy supervision, intercalated P.
vivax infection, and initial parasitaemia were found to be associated with number of days to
recrudescent infection P <0.1, in linear regression analyses, including a single risk factor and
adjusting for age, weight and study period (see Table 3 column Univariable). These
variables remained associated at the nominal level of significance of 0.1, in a linear
regression analysis including all risk factors and adjusting for age, weight and study period
(see Table 3 column Multivariable). The estimated ratio of geometric means for the
statistically, significantly associated, risk factors obtained from the multivariable analysis
suggests an increase in the geometric mean number of days to recrudescent infection of 1.28fold (95 % CI: 1.09-1.51) for women treated with artemisinin based therapy compared to
non-artemisinin based therapy; and 2.19-fold (95 % CI: 1.77-2.72) in those women with an
intercalated P. vivax infection compared to those who did not have intercalated P. vivax. A
decrease in the geometric mean number of days to recrudescent infection of 0.83-fold (95 %
CI: 0.73-0.95) was observed for women whose treatment was incompletely supervised
compared to completely supervised; and 0.98-fold (95 % CI: 0.96-0.99) for each doubling in
the baseline parasitaemia.

Table 3 The estimated ratio of the geometric mean number of days to recrudescence for each risk factor derived from linear regression analyses
of 428 Plasmodium falciparum episodes among 354 pregnant women
Risk factor

Univariablea
Nb
93
256
49
300
232
88
86
253
28
321
349
349
327

Multivariablea
Nb
79
212
38
253
211
80
81
210
25
266
291
291
291

Categories
Ratio of geometric means (95 % CI) [P-value]
Ratio of geometric means (95 % CI) [P-value]
Primiparous
0.99 (0.86, 1.14) [0.890]
1.03 (0.89, 1.19) [0.698]
Parity
Multiparous
1c
1
Artemisinin based
1.28 (1.09, 1.51) [0.003]
1.24 (1.07, 1.43) [0.003]
Anti-malarial therapy
Non-artemisinin based
1
1
Symptomatic
0.89 (0.77, 1.03) [0.120]
0.94 (0.80, 1.11) [0.459]
Symptoms
Asymptomatic
1
1
Unsupervised
0.83 (0.73, 0.94) [0.004]
0.83 (0.73, 0.95) [0.006]
Therapy supervision
Supervised
1
1
Yes
2.19 (1.77, 2.72) [<0.001]
2.19 (1.76, 2.73) [<0.001]
Intercalated P. vivax
No
1
1
EGA (weeks)
349
1.00 (0.99, 1.00) [0.217]
1.00 (0.99, 1.00) [0.210]
Parasitaemia (m/L)d
349
0.98 (0.97, 1.00) [0.079]
0.98 (0.96, 0.99) [0.014]
Haematocrit (%)
327
1.00 (0.99, 1.01) [0.618]
1.00 (0.99, 1.01) [0.654]
Combo, combination; Mono, monotherapy
a
Outcome is time to recrudescent infection and estimates are adjusted for age, weight and study period. Univariable, linear model included a single risk factor, age, weight and study period;
Multivariable, linear model included all risk factors, age, weight and study period
b
The total number of episodes (N) included in each analysis may differ from 428 due to missing risk factor data
c
Indicates reference group
d
Parasitaemia was log2 transformed. Estimate interpreted as a 0.98-fold decrease in geometric mean time to recrudescent Pf infection(s) for a doubling in parasitaemia

Discussion
This study reports the largest, single-site, longitudinal population study of PCR-genotyped P.
falciparum malaria parasitaemia in pregnancy.

Prolonged carriage in pregnant women

The interval from treatment of falciparum malaria in pregnancy to PCR-confirmed
recrudescent infection can be prolonged. The maximum observed time in this large dataset
was 126 days. Pregnant women in this study generally received supervised rather than
unsupervised treatment of P. falciparum. Supervising the treatment was associated with an
increased interval to late recrudescence. Several factors contributed to the high rates of
recrudescence. While all women were treated with standard adult dosing of anti-malarials,
pregnancy alters the pharmacokinetic properties of many anti-malarials lowering their plasma
concentrations [17, 18]. It has been hypothesized that artesunate-treated parasites may enter a
state of quiescence, which protects them from the lethal effects of anti-malarials but later
allows recovery and normal growth [30, 31]. Dormancy has also been postulated to occur
with pyrimethamine, atovaquone and proguanil [32, 33]. However, in this cohort,
combination therapy whether artesunate-based or not was associated with a longer time to
recrudescent infection. Immunity to malaria is reduced in pregnancy and the placenta
provides a favourable site for the parasite to sequester where host defence mechanisms are
attenuated [34].
Intercalated P. vivax infection resulted in a significantly lower proportion of recrudescent P.
falciparum episodes than in women who did not have intercalated P. vivax. Intercalated P.
vivax prolonged the time to recurrence of both novel and recrudescent infections and was the
most significant risk factor for prolonged time to recrudescence. Although the chloroquine
inhibitory concentration of 50 % (IC50) for P. falciparum isolates in this area are amongst the
highest reported in the world [35, 36] some isolates are still partially sensitive to chloroquine.
This is consistent with results that demonstrated a non-significant reduction in P. falciparum
in a double-blind, randomized, placebo-controlled, prophylaxis trial of 1,000 pregnant
women in this setting [29]. In addition, P. vivax may activate host-defences which affect P.
falciparum [37]. The two parasites are competitive and provide mutual suppression. A
protective effect of P. vivax has previously been reported in this area: mixed (P. falciparum
and P. vivax) infection was associated with a four-fold reduction in the risk of developing
severe malaria [22].

Pregnancy and anti-malarial efficacy

The proportion of recrudescent infections with quinine and mefloquine monotherapy was
high and these poorly efficacious regimens should no longer be used in this area [6, 7]. Patent
and sub-patent P. falciparum parasitaemia in pregnancy are harmful to the pregnant woman
and the foetus and prompt and efficacious treatment is required [38]. Frequent screening of a
woman during pregnancy will increase the detection of parasitaemia [2]. Treatment of these
episodes reduces but does not eliminate the adverse effects of low birth weight for the foetus
and anaemia for the mother [2], which can only be achieved by prevention.

This study demonstrates that a significant proportion of pregnant women, 29.0 % by day 28,
13.3 % by day 42 and 5.6 % by day 63 of follow-up, have recrudescent infections beyond the
traditional boundaries used for follow-up in anti-malarial efficacy trials. These proportions
are large enough to underestimate significantly true failure rates with shorter periods of
follow-up. Accurate assessment of anti-malarial efficacy in pregnancy requires follow-up to
delivery, failing this, day 63. This is contrary to modelling data from trials in non-pregnant
patients where 4263 days follow-up captures nearly all anti-malarial treatment failures [4].
There are limitations to this analysis: only three drug treatments (quinine monotherapy,
artesunate monotherapy and artesunate clindamycin) accounted for 85 % of the data. In this
setting, with MDR-P. falciparum no trial that includes both pregnant and non-pregnant
women has been conducted. Attempts to pool data on recrudescent P. falciparum infections
for age-matched women of reproductive age treated with the same anti-malarial and same
year of treatment within the population, i.e., artemether-lumefantrine and dihydroartemisininpiperaquine, were unsuccessful.
While there are reasons to suspect that pregnancy per se many predispose to a longer duration
to recrudescence this study is unable to prove it. Only settings with data including prolonged
duration of follow-up; the same regimen for diagnosis and treatment of P. falciparum
infections in pregnant and non-pregnant women which is most often not the case; and
preferably with women matched for age and gravidity would have the potential to elucidate
this suspicion.

Post-treatment prophylactic effect

No effect of anti-malarial drugs was observed on the post-treatment prophylactic effect, i.e.,
the time to novel recurrence however without a group who receives no anti-malarials this
cannot be proven. There was limited power to detect a post-treatment prophylactic effect and
there were few patients who received long-acting anti-malarials and re-infection rates were
very low, so any effect would have been small. Regarding IPT in this area a study conducted
in men followed for nine months in a randomized, placebo controlled IPT trial required
dihydroartemisinin-piperaquine to be provided monthly at full treatment doses (two tablets
twice per day for three days) to be effective [39]. When re-infection rates of P. falciparum are
very low it is difficult to justify the use of IPT as the risk benefit ratio may not be in favour of
providing this drug to all pregnant women. The current annual incidence of P. falciparum in
pregnancy in the area is now less than 0.5 infections per woman per year [40].

Conclusion
The interval to recrudescence of falciparum malaria in pregnancy can be prolonged,
regardless of the anti-malarial used for treatment. In areas where intercalated P. vivax occurs
and receives treatment, time to recrudescence of P. falciparum can be prolonged. In this area
of low, seasonal malaria transmission, recrudescence occurred after day 42 in approximately
15 % and after day 63 in 5 %, of pregnant women. Accurate characterization of drug efficacy
in pregnancy requires follow-up to delivery or day 63, whichever occurs last.

Competing interests
RM is on the Editorial Board of Malaria Journal. The other authors have declared that they
have no competing interests.

Authors contributions
NL, MI, RM, UL, SZ, NJW, and FN developed the analytic plan and performed the analysis.
NL, AB, MP, RM, JV, MR, and MB participated in collecting the data. AB, RM and FN
participated in developing the initial concept of the study. NL, UL, RM, SZ, NJW, and FN
participated in drafting the manuscript and revision. All authors read and approved the final
manuscript.

Acknowledgements
We sincerely thank the pregnant women who followed the antenatal clinics and agreed to
finger-prick sampling. The work would not have been possible without the combined efforts
of the midwives, medics, laboratory, and logistical teams. Julie A Simpson provided welcome
statistical advice. This research was part of the Wellcome Trust Mahidol University Oxford
Tropical Medicine Research Programme, supported by the Wellcome Trust of Great Britain
(Major Overseas ProgrammeThailand Unit Core Grant).

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